Project Summary Spinal dorsal horn interneurons (IN) integrate somatosensory inputs and control their access to spinal projection neurons (PrN) that transmit nociceptive information to supraspinal components of pain pathways. The heterogeneity of dorsal horn neurons, limited knowledge on their connectivity, and lack of in vivo neurophysiological analysis of identified IN currently preclude comprehensive mapping of circuits involved in pain processing. Our long-term goal is to define dorsal horn neuronal networks in vivo, in the context of somatic sensory stimuli with established behavioral outcomes. We propose to establish a platform for multidisciplinary analysis of dorsal horn circuits based on: 1) monosynaptic transfer of non-toxic self- inactivating modified rabies virus (SiR) for targeted expression of fluorescent reporter proteins and Ca2+ indicators; 2) in vivo three-photon Ca2+ imaging of synaptically connected neurons using genetically encoded Ca2+ indicators; 3) CLARITY-based structural analysis; 4) single-cell transcriptional profiling of functionally characterized synaptically connected neurons. For proof-of-concept of this network analysis, the proposed project is focused on spinal PrN and their presynaptic IN. Information on these IN is extremely limited. We will conduct structural and transcriptomic profiling of PrN neurons and their presynaptic IN and examine in vivo their activity in response to sensory stimuli. Our central hypothesis is that this complementary analysis will identify modality-selective subtypes in the population of IN that are presynaptic to PrN. In Specific Aim 1, we will establish the structural connectivity of PrN and their presynaptic IN by labeling them differentially through monosynaptic gene transfer. CLARITY-based immunohistochemistry and volumetric imaging will be used to establish the structural relationship between the labeled neurons and classify them based on previously established morphological and neurochemical criteria. In Specific Aim 2, we will define responses of PrN and their presynaptic IN to innocuous and noxious thermal and mechanical sensory stimuli using in vivo three- photon Ca2+ imaging. Following in vivo imaging, we will use CLARITY methodology for morphological and neurochemical analysis of the functionally characterized neurons. In Specific Aim 3, we will characterize PrN and their presynaptic IN based on single-cell transcriptional profiling by fluorescently labeling their nuclei through monosynaptic gene transfer and conducting single-nucleus transcriptomic analysis. In addition, we will carry out proof-of-concept experiments for in vivo genetic tagging of neurons following three-photon Ca2+ imaging to correlate single-cell transcriptomes with physiological response properties. Impact: The proposed project will establish a new approach for integrated structural, transcriptional and functional mapping of dorsal horn microcircuits that will address critical gaps in our understanding of spinal pain processing. Monosynaptic tracing of different subsets of dorsal horn neurons in future studies will allow us to systematically and comprehensively explore connectivity in dorsal horn networks.